PROJECT 2 ? CHEMICAL BIOLOGY OF DNA DEAMINASES IN BREAST CANCER ABSTRACT APOBEC enzymes are single-stranded DNA cytosine-to-uracil deaminases that normally protect cells from viral infections. However, APOBEC3B (A3B) has been implicated in mutations in breast cancer that drive tumor evolution and contribute to the development of drug resistance and, ultimately, therapy failure. A3B is overexpressed in over half of all estrogen receptor (ER)-positive breast tumors, the most common type, and is associated with poor overall survival. Our Program has shown that inhibition of A3B-mediated tumor evolution improves therapy outcomes in a mouse model of ER-positive breast cancer. Our Program?s unifying hypothesis is that A3B inhibition will prevent a large proportion of new mutations in ER-positive breast cancer, thereby improving the durability of current treatments and resulting in better overall outcomes. To address this hypothesis our Program is focused on understanding the biology of A3B in breast cancer cells (Project 1); developing innovative nucleic acid probes to molecularly characterize how A3B engages DNA substrates and small molecules to inhibit A3B-catalyzed breast cancer mutations (Project 2); and generating A3B x-ray structures with nucleic acids, small molecules, and protein ligands to understand the structural basis of A3B- mediated DNA mutagenesis and its inhibition to enable development of therapeutic compounds (Project 3). These activities will be supported by Service Cores for administration (Core A), animal models of A3B-driven breast cancer (Core B), computational chemistry and biophysics (Core C), and protein and antibody production (Core D). Project 2 ? Chemical Biology of DNA Deaminases in Breast Cancer will lead the chemical probe discovery efforts by 1) synthesizing complex nucleic acid ligands for A3B to characterize how A3B discriminates 2-deoxycytidine from other nucleotides and to understand which nucleic acid features enable A3B to deaminate discrete DNA sequences, including its overall preference for binding DNA versus RNA; and 2) using complimentary technologies and approaches to develop first-in-class small molecule inhibitors of A3B that will be used in mechanistic cellular assays of A3B-driven breast cancer mutation (Project 1), structural biology studies to annotate A3B-ligand binding (Project 3), and therapeutic utility experiments in animal models of breast cancer (Core B). Our studies will be enabled by critical collaborations involving computational ligand design (Core C) and access to high-quality biological reagents for assays (Core D), and our advances will position our novel compounds for future therapeutic development. Potent, selective chemical probes of A3B with in vivo activity, as well as novel assays, are the major anticipated deliverables of Project 2. As such, Project 2 will be the center of chemical innovation for the Program, accelerating all Projects and contributing to the achievement of our overall research objectives.